小麦茎秆果聚糖关键水解酶基因的克隆与功能验证

The accumulation and remobilization of water-soluble carbohydrates (WSC) in wheat stem are a key factor influencing on grain yield. Fructan is a major component of WSC in wheat stem. The mobilization of stored WSC requires the hydrolysis of fructans, which is catalyzed by fructan exohydrolases. The activities of 1-exohydrolases (1-FEH) and fructan 6-exohydrolases (6-FEH) are important for fructan remobilization efficiency during mid-to-late grain filling stage, particularly for promoting grain filling rate and yield under drought and heat conditions. In this project, the full-length genomic DNA sequence of 1-FEH and 6-FEH genes will be characterized by homologous cloning and experimental validation. Allelic variants for high fructan metabolism will be identified, and functional markers of 1-FEH and 6-FEH genes will be developed based on the polymorphisms among genotypes. The functional markers will be mapped using a set of Chinese Spring milli-tetrasomic and ditelosomic lines, and a RIL population from the cross of Doumai/Shi 4185. Meanwhile, the WSC and fructan remobilization will be measured in wheat stem of the RILs, and used for quantitative trait loci (QTL) mapping. The genetic effects on WSC and fructan mobilization conferred by the genes 1-FEH and 6-FEH, co-segregating with the functional markers, will be estimated in the RIL population. The genetic relationships between the key hydrolysis genes and main QTLs for WSC and fructan remobilization will be analyzed. A total of 166 wheat varieties from major wheat grown areas in China will be used to validate the association between the functional markers and stem carbohydrate mobilization. The mRNA transcription and protein expression of two hydrolase genes will be analyzed based on the fructan differences among varieties during grain filling stage, which will be valuable for understanding the transferring pathway of fructan remobilization. The function of two hydrolase genes will be confirmed by gene over-expression and RNAi approaches. The results of the program will extend our knowledge on both key genes of fructan remobilization, and accelerate the progress for the improvement of high and stable yield by providing genes, germplasm and molecular markers in wheat breeding.

小麦茎秆碳水化合物（WSC）积累与转运是影响籽粒产量的关键因素，果聚糖是WSC的主要组分，其水解转运对籽粒灌浆和耐旱热胁迫尤为重要。本项目根据果聚糖关键水解酶1-FEH和6-FEH基因序列，发掘控制果聚糖代谢的优异等位变异，结合等位基因的序列差异，开发水解酶基因的功能标记。利用中国春缺体-四体对标记进行定位，并在豆麦/石4185 RIL群体中鉴定1-FEH和6-FEH基因的位置和遗传效应。以166份主产麦区品种为材料，分析水解酶基因的等位变异与WSC和果聚糖转运量的关系，验证功能标记的有效性。以果聚糖转运量高低差异的品种各4份，分析茎秆生长发育中后期1-FEH和6-FEH基因的转录表达，明确水解酶基因表达调控果聚糖转运量的信息。通过转基因过表达和RNAi等手段，解析这两个基因的功能和利用价值。本研究将推进小麦育种在提高干物质转运效率方面的认识，为培育高产稳产耐旱新品种提供基因、种质和标记。

小麦WSC10、WSC20和WSC30遗传力较高，可通过分子标记辅助选择对其高效改良。小麦WSC10、WSC20与TKW相关性较高，可通过提高茎秆WSC10和WSC20来提高TKW。GWAS鉴定到62个稳定茎秆WSC含量关联位点，其分布在除5D染色体外的20条染色体上，覆盖36个WSC10、24个WSC20和19个WSC30位点。发现过去70年，黄淮麦区小麦品种茎秆WSC含量和TKW逐渐提高，品种所含茎秆WSC含量优异等位基因数量逐渐增多。定位到5个WSC10、2个WSC20和1个WSC30位点，其中6个位点优异等位基因来自中麦895，3个位点与GWAS定位结果一致。鉴定到6A染色体上1-FEH基因三种新单倍型，针对其第一外显子一个错义突变开发了KASP标记1-FEH-AM1，可有效用于提高灌浆期果聚糖含量的分子育种。对多个材料测序发现，6B和6D染色体上1-FEH基因转录起始点至转录终止点序列在黄淮麦区小麦材料中缺乏多态性。克隆出6-FEH全基因序列目标片段，发现6-FEH包括6个外显子、5个内含子及5”和3”端的非编码区。对5个重要茎秆WSC含量和TKW多效位点开发了KASP标记，可有效用于小麦茎秆WSC含量和TKW分子育种。开发了1-FEH w1功能基因KASP标记1-FEH-AM1，可有效用于小麦WSC10分子辅助选择改良。